Seed Yield Regulator (SYR)
There is a continuous need to find new seed yield enhancement genes and several approaches have been used so far, for example through manipulation of plant hormone levels (WO 03/050287), through manipulation of the cell cycle (WO 2005/061702), through manipulation of genes involved in salt stress response (WO 2004/058980) amongst other strategies.
SYR is a new protein that has hitherto not been characterised. SYR shows some homology (around 48% sequence identity on the DNA level, around 45% on the protein level) to an Arabidopsis protein named ARGOS (Hu et al., Plant Cell 15, 1951-1961, 2003; US 2005/0108793). Hu et al. postulated that ARGOS is a protein of unique function and is encoded by a single gene. The major phenotypes of ARGOS overexpression in Arabidopsis are increased leafy biomass and delayed flowering.
FG-GAP
FG-GAP proteins are putative transmembrane proteins. They are characterised by the presence of one or more FG-GAP domains (Pfam accession number PF01839) and by the presence of an N-terminal signal peptide and a transmembrane domain in the C-terminal half of the protein.
One such protein, DEX1, was isolated from Arabidopsis and was reported to play a role during pollen development (Paxson-Sowders et al. Plant Physiol. 127, 1739-1749, 2001). Dex1 mutant plants were shown to be defective in pollen wall pattern formation. The DEX1 gene encodes an 896-amino acid protein that is predicted to localize to the plasma membrane, with residues 1 through to 860 being located outside of the cell, residues 880 through to 895 on the cytoplasmic side of the membrane, and amino acids 861 through to 879 representing a potential membrane-spanning domain. Twelve potential N-glycosylation sites are present in DEX1. Therefore, the protein has the potential to be heavily modified and interact with various components of the cell wall. DEX1 shows the greatest sequence similarity to a hemolysin-like protein from V. cholerae, whereas an approximately 200-amino acid segment of DEX1 (amino acids 439643) also shows limited similarity to the calcium-binding domain of alpha-integrins. In this region are at least two sets of putative calcium-binding ligands that are also present in a predicted Arabidopsis calmodulin protein (AC009853). Therefore, it appears that DEX1 may be a calcium-binding protein. DEX1 appears to be a unique plant protein; homologs are not present in bacteria, fungi, or animals.
The alterations observed in dex1 plants, as well as the predicted structure of DEX1, raise several possibilities for the role of the protein in pollen wall formation (Paxson-Sowders et al., 2001):                (a) DEX1 could be a linker protein. It may associate with the microspore membrane and participate in attaching either the primexine or sporopollenin to the plasma membrane. Absence of the protein from the microspore surface could result in structural alterations in the primexine. The numerous potential N-glycosylation sites are consistent with attachment of DEX1 to the callose wall, the intine, or both.        (b) DEX1 may be a component of the primexine matrix and play a role in the initial polymerization of the primexine. Changes in Ca+2 ion concentrations appear to be important for pollen wall synthesis; beta-glucan synthase is activated by micromolar concentrations of Ca+2 during callose wall formation.        
(c) DEX1 could be part of the rough ER and be involved in processing and/or transport of primexine precursors to the membrane. The delayed appearance and general alterations in the primexine are consistent with a general absence of primexine precursors. The primexine matrix is initially composed of polysaccharides, proteins, and cellulose, followed by the incorporation of more resistant materials. Therefore, DEX1 may participate in the formation or transport of any number of different components.
CYP90B
Brassinosteroids (BRs) are a class of plant hormones that are important for promoting plant growth, division and development. The term BR collectively refers to more than forty naturally occurring poly-hydroxylated sterol derivatives, with structural similarity to animal steroid hormones. Among these, brassinolide has been shown to be the most biologically active (for review, Clouse (2002) Brassinosteroids. The Arabidopsis Book: 1-23).
The BR biosynthetic pathway has been elucidated using biochemical and mutational analyses. BRs are synthesized via at least two branched biochemical pathways starting from the same initial precursor, campesterol (Fujioka et al. (1997) Physiol Plant 100:710-715). The discovered BR biosynthesis genes have been found to encode mostly cytochrome P450 monooxygenases (CYP) (Bishop and Yokota (2001) Plant Cell Physiol 42:114-120). CYP superfamily of enzymes catalyses the oxidation of many chemicals, and in the present case more specifically catalyse essential oxidative reactions in the biosynthesis of BRs. One of the important steps identified consists in the hydroxylation of the steroid side chain of BR intermediates campestanol and 6-oxocampestanol to form 6-deoxocathasterone and cathasterone respectively. These two parallel oxidative steps are also collectively called the early steroid C-22 alpha-hydroxylation step (Choe et al. (1998) Plant Cell 10: 231-243). In Arabidopsis, a specific CYP enzyme, CYP90B1 or DWF4, performs this step (for general reference on plant CYP nomenclature, Nelson et al. (2004) Plant Phys 135: 756-772).
Arabidopsis mutant plants lacking steroid 22 alpha hydroxylase activity due insertion of a T-DNA in the DWF4 locus displayed a dwarfed phenotype due to lack of cell elongation (Choe et al. (1998) Plant Cell 10: 231-243). Biochemical feeding studies with BR biosynthesis intermediates showed that all of the downstream compounds rescued the phenotype, whereas the known precursors failed to do so.
Transgenic Arabidopsis and tobacco plants, both dicotyledonous, were generated that ectopically overexpressed an Arabidopsis DWF4 genomic fragment, using the cauliflower mosaic virus 35S promoter (Choe et al. (2001) Plant J 26(6): 573-582). Phenotypic characterisation of the plants showed that the hypocotyl length, plant height at maturity, total number of branches and total number of seeds were increased in the transgenics compared to control plants. Choe et al. found that the increased seed production was due to a greater number of seeds per plant, seed size increase being within the range of standard deviation. These experiments are further described in WO00/47715.
U.S. Pat. No. 6,545,200 relates to isolated nucleic acid fragments encoding sterol biosynthetic genes, and more specifically claims a nucleotide sequence encoding a polypeptide having C-8,7 sterol isomerase activity. Partial nucleotides sequences encoding DWF4 are disclosed.
US 2004/0060079 relates to a method of producing a modified monocotyledonous plant having a desired trait. An example is provided in which the rice DWF4-encoding nucleotide sequence (referred to either OsDWF4 or CYP90B2) is placed under the control of a constitutive promoter, the rice actin promoter. Fourteen of the thirty-six transgenic rice plants expressing the chimeric construct show an increased number of grains per spike as compared to non-transformed control plants. According to the inventors, the yield increase in the transgenics compared to the wild types is due to an increase in total number of seeds, as no significant difference is found in the “weight of 10 grains”. CDC27
Depending on the end use, the modification of certain yield traits may be favoured over others. For example for applications such as forage or wood production, or bio-fuel resource, an increase in the leafy parts of a plant may be desirable, and for applications such as flour, starch or oil production, an increase in seed parameters may be particularly desirable. Even within the seed parameters, some may be favoured over others, depending on the application. Various mechanisms may contribute to increasing seed yield, whether that is in the form of increased seed size or increased seed number. One such mechanism is the cell cycle.
Progression through the cell cycle is fundamental to the growth and development of all multicellular organisms and is crucial to cell proliferation. The major components of the cell cycle are highly conserved in yeast, mammals, and plants. The cell cycle is typically divided into the following sequential phases: G0-G1-S-G2-M. DNA replication or synthesis generally takes place during the 8 phase (“S” is for DNA synthesis) and mitotic segregation of the chromosomes occurs during the M phase (the “M” is for mitosis), with intervening gap phases, G1 (during which cells grow before DNA replication) and G2 (a period after DNA replication during which the cell prepares for division). Cell division is completed after cytokinesis, the last step of the M phase. Cells that have exited the cell cycle and that have become quiescent are said to be in the G0 phase. Cells in this phase can be stimulated to renter the cell cycle at the G1 phase. The “G” in G1, G2 and G0 stands for “gap” Completion of the cell cycle process allows each daughter cell during cell division to receive a full copy of the parental genome.
Cell division is controlled by two principal cell cycle events, namely initiation of DNA synthesis and initiation of mitosis. Each transition to each of these key events is controlled by a checkpoint represented by specific protein complexes (involved in DNA replication and division). The expression of genes necessary for DNA synthesis at the G1/S boundary is regulated by the E2F family of transcription factors in mammals and plant cells (La Thangue, 1994; Muller et al., 2001; De Veylder et al., 2002). Entry into the cell cycle is regulated/triggered by an E2F/Rb complex that integrates signals and allows activation of transcription of cell cycle genes. The transition between the different phases of the cell cycle, and therefore progression through the cell cycle, is driven by the formation and activation of different heterodimeric serine/threonine protein kinases, generally referred to as cyclin-dependent kinases (CDKs). A prerequisite for activity of these kinases is the physical association with a specific cyclin, the timing of activation being largely dependent upon cyclin expression. Cyclin-binding induces conformational changes in the N-terminal lobe of the associating CDK and contributes to the localisation and substrate specificity of the complex. Monomeric CDKs are activated when they are associated with cyclins and thus have a kinase activity. Cyclin protein levels fluctuate in the cell cycle and therefore represent a major factor in determining timing of CDK activation. The periodic activation of these complexes containing cyclins and CDK during cell cycle mediates the temporal regulation of cell cycle transitions (checkpoints).
Mechanisms exist to ensure that DNA replication occurs only once during the cell cycle. For example, CDC16, CDC23 and CDC27 proteins are part of a high molecular weight complex known as the anaphase promoting complex (APC) or cyclosome, (see Romanowski and Madine, Trends in Cell Biology 6, 184-188, 1996, and Wuarin and Nurse, Cell 85, 785787 (1996). The complex in yeast is composed of at least eight proteins, the TPR-(tetratrico peptide repeat) containing proteins CDC16, CDC23 and CDC27, and five other subunits named APC1, APC2, APC4, APC5 and APC7 (Peters et al. 1996, Science 274, 1199-1201). The APC targets its substrates for proteolytic degradation by catalyzing the ligation of ubiquitin molecules to these substrates. APC-dependent proteolysis is required for the separation of the sister chromatids at meta- to anaphase transition and for the final exit from mitosis. Among the APC-substrates are the anaphase inhibitor protein Pds1p and mitotic cyclins such as cyclin B, respectively (Ciosk et al. 1998, Cell 93, 1067-1076; Cohen-Fix et al. 1996, Genes Dev 10, 3081-3093; Sudakin et al. 1995, Mol Biol Cell 6, 185-198; Jorgensen et al. 1998, Mol Cell Biol 18, 468-476; Townsley and Ruderman 1998, Trends Cell Biol 8, 238-244). To become active as an ubiquitin-ligase, at least CDC16, CDC23 and CDC27 need to be phosphorylated in the M-phase (Ollendorf and Donoghue 1997, J Biol Chem 272, 32011-32018). Activated APC persists throughout G1 of the subsequent cell cycle to prevent premature appearance of B-type cyclins, which would result in an uncontrolled entry into the S-phase (Imiger and Nasmyth 1997, J Cell Sci 110, 1523-1531). It has been demonstrated in yeast that mutations in either of at least two of the APC components, CDC16 and CDC27, can result in DNA overreplication without intervening passages through M-phases (Heichman and Roberts 1996, Cell 85, 39-48). This process of replication of nuclear DNA without subsequent mitosis and cell division is called DNA endoreduplication, and leads to increased cell size.
CDC16, CDC23 and CDC27 all are tetratrico peptide repeat (TPR; 34 amino acids long) containing proteins. A suggested minimal consensus sequence of the TPR motif is as follows: X3-W-X2-L-G-X2-Y-X8-A-X3-F-X2-A-X4-P-X2, (SEQ ID NO: 286) where X is any amino acid (Lamb et al. 1994, EMBO J. 13, 4321-4328). The consensus residues can exhibit significant degeneracy and little or no homology is present in non-consensus residues. It is the hydrophobicity and size of the consensus residues, rather than their identity, that seems to be of importance. TPR motifs are present in a wide variety of proteins functional in yeast and higher eukaryotes in mitosis (including the APC protein components CDC16, CDC23 and CDC27), transcription, splicing, protein import and neurogenesis (Goebl and Yanagida 1991, Trends Biochem Sci 16, 173-177). The TPR forms an α-helical structure; tandem repeats organize into a superhelical structure ideally suited as interfaces for protein recognition (Groves and Barford 1999, Curr Opin Struct Biol 9, 383-389). Within the α-helix, two amphipathic domains are usually present, one at the NH2 terminal region and the other near the COOH terminal region (Sikorski et al. 1990, Cell 60, 307-317).
CDC27 (also known as Hobbit; others names include CDC27, BimA, Nuc2 or makos) has been isolated from various organisms, including Aspergillus nidulans, yeast, drosophila, human and various plants (such as Arabidopsis thaliana and Otyza sativa). The gene encoding CDC27 is present as a single copy in most genomes, but two copies may exceptionally be found within the same genome, for example in Arabidopsis thaliana. The two genes encoding CDC27 proteins have been named CDC27A and CDC27B (MIPS references At3g16320 and At2g20000 respectively).
Published International Patent Application, WO01/02430 describes CDC27A (CDC27A1 and CDC27A2) and CDC27B sequences. Also described in this document is a truncated CDC27B amino add sequence in which 161 amino acids are missing from the NH2 terminal region. Reference is made in this document to GenBank accession number AC006081 for the CDC27B gene encoding a CDC27B polypeptide truncated at the NH2 terminal region. The document reports the NH2 terminal region to be conserved in CDC27 homologues of different origin. The CDC27 sequences mentioned in WO01/02430 are described to be useful in modifying endoreduplication.
DNA endoreduplication occurs naturally in flowering plants, for example during seed development. DNA endoreduplication leads to enlarged nuclei with elevated DNA content It has been suggested that the increased DNA content during endoreduplication may provide for increased gene expression during endosperm development and kernel filling, since it coincides with increased enzyme activity and protein accumulation at this time (Kowles et al., (1992) Genet. Eng. 14:65-88). In cereal species, the cellular endosperm stores the reserves of the seed during a phase marked by endoreduplication. The magnitude of DNA endoreduplication is highly correlated with endosperm fresh weight, which implies an important role of DNA endoreduplication in the determination of endosperm mass (Engelen-Eigles et al. (2000) Plant Cell Environ. 23:657-663). In maize for example, the endosperm makes up 70 to 90% of kernel mass; thus, factors that mediate endosperm development to a great extent also determine grain yield of maize, via individual seed weight. Increased endoreduplication is therefore typically indicative of increased seed biomass but is in no way related to increased seed number.
AT-Hook Transcription Factor
An AT-hook domain is found in polypeptides belonging to a family of transcription factors associated with Chromatin remodeling. The AT-hook motif is made up of 13 or so (sometimes about 9) amino acids which participate in DNA binding and which have a preference for A/T rich regions. In Arabidopsis there are at least 34 proteins containing AT-hook domains. These proteins share homology along most of the sequence, with the AT-hook domain being a particularly highly conserved region.
International Patent application WO 2005/030966 describes several plant transcription factors comprising AT-hook domains and the use of these transcription factors to produce plants having increased biomass and increased stress tolerance. The application concerns members of the G1073 lade of transcription factors and states that, “Use of tissue-specific or inducible promoters mitigates undesirable morphological effects that may be associated with constitutive overexpression of G1073 clade members (e.g., when increased size is undesirable).” The data provided in this application relate to dicotyledonous plants.
In contrast to these teachings, it has now been found that expression in a monocotyledonous (monocot) plant of a polynucleic acid encoding an AT-hook transcription factor comprising a DUF296 domain (which includes members of lade G1073), gives plants having little or no increase in biomass compared with suitable control plants, regardless of whether that expression is driven by a constitutive promoter or in a tissue-specific manner. This suggests that teachings concerning expression of such transcription factors in dicots may not be so readily applicable to monocots. It has also now been found that the extent or nature of any increase in seed yield obtained is dependent upon the tissue-specific promoter used.
DOF Transcription Factors
Dof domain proteins are plant-specific transcription factors with a highly conserved DNA-binding domain with a single C2-C2 zinc finger. During the past decade, numerous Dof domain proteins have been identified in both monocots and dicots including maize, barley, wheat, rice, tobacco, Arabidopsis, pumpkin, potato and pea. Dof domain proteins have been shown to function as transcriptional activators or repressors in diverse plant-specific biological processes.
Cyclin Dependent Kinase Inhibitors (CKI)
The ability to increase plant seed yield, whether through seed number, seed biomass, seed development, seed filling or any other seed-related trait would have many applications in agriculture, and even many non-agricultural uses such as in the biotechnological production of substances such as pharmaceuticals, antibodies or vaccines. One approach to increasing seed yield in plants may be through modification of the inherent growth mechanisms of a plant.
The inherent growth mechanisms of a plant reside in a highly ordered sequence of events collectively known as the ‘cell cycle’. Progression through the cell cycle is fundamental to the growth and development of all multi-cellular organisms and is crucial to cell proliferation. The major components of the cell cycle are highly conserved in yeast, mammals, and plants. The cell cycle is typically divided into the following sequential phases: G0-G1-S-G2-M. DNA replication or synthesis generally takes place during the S phase (“S” is for DNA synthesis) and mitotic segregation of the chromosomes occurs during the M phase (the “M” is for mitosis), with intervening gap phases, G1 (during which cells grow before DNA replication) and G2 (a period after DNA replication during which the cell prepares for division). Cell division is completed after cytokinesis, the last step of the M phase. Cells that have exited the cell cycle and that have become quiescent are said to be in the G0 phase. Cells in this phase can be stimulated to renter the cell cycle at the G1 phase. The “G” in G1, G2 and G0 stands for “gap”. Completion of the cell cycle process allows each daughter cell during cell division to receive a full copy of the parental genome.
Cell division is controlled by two principal cell cycle events, namely initiation of DNA synthesis and initiation of mitosis. Each transition to each of these key events is controlled by a checkpoint represented by specific protein complexes (involved in DNA replication and division). The expression of genes necessary for DNA synthesis at the G1/S boundary is regulated by the E2F family of transcription factors in mammals and plant cells (La Thangue, 1994; Muller et al., 2001; De Veylder et al., 2002). Entry into the cell cycle is regulated/triggered by an E2F/Rb complex that integrates signals and allows activation of transcription of cell cycle genes. The transition between the different phases of the cell cycle, and therefore progression through the cell cycle, is driven by the formation and activation of different heterodimeric serine/threonine protein kinases, generally referred to as cyclin-dependent kinases (CDKs). A prerequisite for activity of these kinases is the physical association with a specific cyclin, the timing of activation being largely dependent upon cyclin expression. Cyclin binding induces conformational changes in the N-terminal lobe of the associating CDK and contributes to the localisation and substrate specificity of the complex. Monomeric CDKs are activated when they are associated with cyclins and thus have kinase activity. Cyclin protein levels usually fluctuate in the cell cycle and therefore represent a major factor in determining timing of CDK activation. The periodic activation of these complexes containing cyclins and CDK during cell cycle mediates the temporal regulation of cell-cycle transitions (checkpoints). Other factors regulating CDK activity include cyclin dependent kinase inhibitors (CKIs or ICKs, KIPs, CIPs, INKs), CDK activating kinases (CAKs), a CDK phosphatase (Cdc25) and a CDK subunit (CKS) (Mironov et al. 1999; Reed 1996).
The existence of an inhibitor of mitotic CDKs was inferred from experiments with endosperm of maize seed (Grafi and Larkins (1995) Science 269, 1262-1264). Since then, several CKIs have been identified in various plant species, such as Arabidopsis (Wang et al. (1997) Nature 386(6624): 451-2; De Veylder et al. (2001) Plant Cell 13: 1653-1668; Lui et al. (2000) Plant J 21: 379-385), tobacco (Jasinski et al. (2002) Plant Physiol 2002 130(4): 871-82), Chenopodium rubrum (Fountain et al. (1999) Plant Phys 120: 339) or corn (Coelho et al. (2005) Plant Physiol 138: 2323-2336). The encoded proteins are characterized by a stretch of approximately 45 carboxy-terminal amino acids showing homology to the amino-terminal cyclin/Cdk binding domain of animal CKIs of the p21Clp1/p27Klp2/p57Klp2-types. Outside this carboxy-terminal region, plant CKIs show little homology.
Published International patent application WO 2005/007829 in the name of Monsanto Technology LLC describes various isolated nucleic acid molecules encoding polypeptides having cyclin dependent kinase inhibitor activity.
Published International patent applications, WO 02/28893 and WO 99/14331, both in the name of CropDesign N.V., describe various plant cyclin dependent kinase inhibitors. The use of these inhibitors to increase yield is mentioned in these applications.